NAADP as a second messenger: neither CD38 nor base-exchange reaction are necessary for in vivo generation of NAADP in myometrial cells

2007 ◽  
Vol 292 (1) ◽  
pp. C227-C239 ◽  
Author(s):  
Sandra Soares ◽  
Michael Thompson ◽  
Thomas White ◽  
Amir Isbell ◽  
Michiko Yamasaki ◽  
...  
Keyword(s):  
Exchange Reaction ◽  
Mammalian Cells ◽  
Second Messenger ◽  
Base Exchange ◽  
Myometrial Cells ◽  
Intracellular Ca ◽  
Messenger System

Nicotinic acid adenine dinucleotide phosphate (NAADP) has recently been shown to act as a second messenger controlling intracellular Ca2+ responses in mammalian cells. Many questions remain regarding this signaling pathway, including the role of the ryanodine receptor (RyR) in NAADP-induced Ca2+ transients. Furthermore, the exact metabolic pathway responsible for the synthesis of NAADP in vivo has not been determined. Here, we demonstrate that the NAADP mediated Ca2+ release system is present in human myometrial cells. We also demonstrate that human myometrial cells use the NAADP second messenger system to generate intracellular Ca2+ transients in response to histamine. It has been proposed in the past that the NAADP system in mammalian cells is dependent on the presence of functional RyRs. Here, we observed that the histamine-induced Ca2+ transients are dependent on both the NAADP and inositol 1,4,5-trisphosphate signaling pathways but are independent of RyRs. The enzyme CD38 has been shown to catalyze the synthesis of NAADP in vitro by the base-exchange reaction. Furthermore, it has been proposed that this enzyme is responsible for the intracellular generation of NAADP in vivo. Using CD38 knockout mice, we observed that both the basal and histamine stimulated levels of NAADP are independent of CD38 and the base-exchange reaction. Our group is the first to demonstrate that NAADP is a second messenger for histamine-elicited Ca2+ transients in human myometrial cells. Furthermore, the NAADP mediated mechanism in mammalian cells can be independent of RyRs and CD38. Our data provides novel insights into the understanding of the mechanism of action and metabolism of this new second messenger system.

Paradoxical effect of salbutamol in a model of acute organophosphates intoxication in guinea pigs: role of substance P release

2007 ◽  
Vol 292 (4) ◽  
pp. L915-L923 ◽  
Author(s):  
Jaime Chávez ◽  
Patricia Segura ◽  
Mario H. Vargas ◽  
José Luis Arreola ◽  
Edgar Flores-Soto ◽  
...  
Keyword(s):  
Substance P ◽  
Guinea Pigs ◽  
Smooth Muscle Contraction ◽  
In Vivo Experiments ◽  
Intracellular Ca ◽  
Neurokinin 1 ◽  
Substance P Release

Organophosphates induce bronchoobstruction in guinea pigs, and salbutamol only transiently reverses this effect, suggesting that it triggers additional obstructive mechanisms. To further explore this phenomenon, in vivo (barometric plethysmography) and in vitro (organ baths, including ACh and substance P concentration measurement by HPLC and immunoassay, respectively; intracellular Ca2+ measurement in single myocytes) experiments were performed. In in vivo experiments, parathion caused a progressive bronchoobstruction until a plateau was reached. Administration of salbutamol during this plateau decreased bronchoobstruction up to 22% in the first 5 min, but thereafter airway obstruction rose again as to reach the same intensity as before salbutamol. Aminophylline caused a sustained decrement (71%) of the parathion-induced bronchoobstruction. In in vitro studies, paraoxon produced a sustained contraction of tracheal rings, which was fully blocked by atropine but not by TTX, ω-conotoxin (CTX), or epithelium removal. During the paraoxon-induced contraction, salbutamol caused a temporary relaxation of ∼50%, followed by a partial recontraction. This paradoxical recontraction was avoided by the M2- or neurokinin-1 (NK1)-receptor antagonists (methoctramine or AF-DX 116, and L-732138, respectively), accompanied by a long-lasting relaxation. Forskolin caused full relaxation of the paraoxon response. Substance P and, to a lesser extent, ACh released from tracheal rings during 60-min incubation with paraoxon or physostigmine, respectively, were significantly increased when salbutamol was administered in the second half of this period. In myocytes, paraoxon did not produce any change in the intracellular Ca2+ basal levels. Our results suggested that: 1) organophosphates caused smooth muscle contraction by accumulation of ACh released through a TTX- and CTX-resistant mechanism; 2) during such contraction, salbutamol relaxation is functionally antagonized by the stimulation of M2 receptors; and 3) after this transient salbutamol-induced relaxation, a paradoxical contraction ensues due to the subsequent release of substance P.

scholarly journals Functional genetic encoding of sulfotyrosine in mammalian cells

2020 ◽  
Vol 11 (1) ◽  
Author(s):  
Xinyuan He ◽  
Yan Chen ◽  
Daisy Guiza Beltran ◽  
Maia Kelly ◽  
Bin Ma ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Biochemical Characterization ◽  
Trna Synthetase ◽  
Functional Verification ◽  
Cellular Processes ◽  
Genetic Encoding ◽  
Efficient Expression

Abstract Protein tyrosine O-sulfation (PTS) plays a crucial role in extracellular biomolecular interactions that dictate various cellular processes. It also involves in the development of many human diseases. Regardless of recent progress, our current understanding of PTS is still in its infancy. To promote and facilitate relevant studies, a generally applicable method is needed to enable efficient expression of sulfoproteins with defined sulfation sites in live mammalian cells. Here we report the engineering, in vitro biochemical characterization, structural study, and in vivo functional verification of a tyrosyl-tRNA synthetase mutant for the genetic encoding of sulfotyrosine in mammalian cells. We further apply this chemical biology tool to cell-based studies on the role of a sulfation site in the activation of chemokine receptor CXCR4 by its ligand. Our work will not only facilitate cellular studies of PTS, but also paves the way for economical production of sulfated proteins as therapeutic agents in mammalian systems.

scholarly journals Streptolysin-induced endoplasmic reticulum stress promotes group A streptococcal in vivo biofilm formation and necrotizing fasciitis

2017 ◽  
Author(s):  
Anuradha Vajjala ◽  
Debabrata Biswas ◽  
Kelvin Kian Long Chong ◽  
Wei Hong Tay ◽  
Emanuel Hanski ◽  
...  
Keyword(s):  
Soft Tissue ◽  
Er Stress ◽  
Necrotizing Fasciitis ◽  
Biofilm Formation ◽  
Mammalian Cells ◽  
Chronic Infections ◽  
Group A

AbstractGroup A Streptococcus (GAS) is a human pathogen that causes infections ranging from mild to fulminant and life-threatening. Biofilms have been implicated in acute GAS soft-tissue infections such as necrotizing fasciitis (NF). However, most in vitro models used to study GAS biofilms have been designed to mimic chronic infections and insufficiently recapitulate in vivo conditions and the host-pathogen interactions that might influence biofilm formation. Here we establish and characterize an in vitro model of GAS biofilm development on mammalian cells that simulates microcolony formation observed in a murine model of human NF. We show that on mammalian cells, GAS forms dense aggregates that display hallmark biofilm characteristics including a three-dimensional architecture and enhanced tolerance to antibiotics. In contrast to abiotic-grown biofilms, host-associated biofilms require the expression of secreted GAS streptolysins O and S (SLO, SLS) resulting in the release of a host-associated biofilm promoting-factor(s). Supernatants from GAS-infected mammalian cells or from cells treated with endoplasmic reticulum (ER) stressors restore biofilm formation to an SLO and SLS null mutant that is otherwise attenuated in biofilm formation on cells, together suggesting a role for streptolysin-induced ER stress in this process. In an in vivo mouse model, the streptolysin-null mutant is attenuated in both microcolony formation and bacterial spread, but pre-treatment of softtissue with an ER-stressor restores the ability of the mutant to form wild type like microcolonies that disseminate throughout the soft tissue. Taken together, we have identified a new role of streptolysin-driven ER stress in GAS biofilm formation and NF disease progression.Significance StatementAlthough it is well-accepted that bacterial biofilms are associated with many chronic infections, little is known about the mechanisms by which group A Streptococcus (GAS) biofilms contribute to acute soft tissue-invasive diseases like necrotizing fasciitis (NF). In this study, we establish a physiologically relevant in vitro model to study GAS biofilm formation on mammalian cells and validate our findings in a mouse model that mimics human NF. This study demonstrates a novel role of GAS streptolysin-mediated ER stress in the development and spread of GAS biofilms in acute softtissue infections. We also show that biofilm formation depends on the release of a host-associated factor that promotes microcolony formation and GAS dissemination in vivo.

scholarly journals Role of proteoglycans and glycosaminoglycans in Duchenne muscular dystrophy

Glycobiology ◽  
2018 ◽  
Vol 29 (2) ◽  
pp. 110-123 ◽  
Author(s):  
Laurino Carmen ◽  
Vadala’ Maria ◽  
Julio Cesar Morales-Medina ◽  
Annamaria Vallelunga ◽  
Beniamino Palmieri ◽  
...  
Keyword(s):  
Duchenne Muscular Dystrophy ◽  
Muscular Dystrophy ◽  
Mammalian Cells ◽  
Muscle Development ◽  
Glycoprotein Complex ◽  
Experimental Therapies ◽  
Dystrophin Protein

Abstract Duchenne muscular dystrophy (DMD) is an inherited fatal X-linked myogenic disorder with a prevalence of 1 in 3500 male live births. It affects voluntary muscles, and heart and breathing muscles. DMD is characterized by continuous degeneration and regeneration cycles resulting in extensive fibrosis and a progressive reduction in muscle mass. Since the identification of a reduction in dystrophin protein as the cause of this disorder, numerous innovative and experimental therapies, focusing on increasing the levels of dystrophin, have been proposed, but the clinical improvement has been unsatisfactory. Dystrophin forms the dystrophin-associated glycoprotein complex and its proteins have been studied as a promising novel therapeutic target to treat DMD. Among these proteins, cell surface glycosaminoglycans (GAGs) are found almost ubiquitously on the surface and in the extracellular matrix (ECM) of mammalian cells. These macromolecules interact with numerous ligands, including ECM constituents, adhesion molecules and growth factors that play a crucial role in muscle development and maintenance. In this article, we have reviewed in vitro, in vivo and clinical studies focused on the functional role of GAGs in the pathophysiology of DMD with the final aim of summarizing the state of the art of GAG dysregulation within the ECM in DMD and discussing future therapeutic perspectives.

scholarly journals The Bacterial Second Messenger Cyclic di-GMP Regulates Brucella Pathogenesis and Leads to Altered Host Immune Response

2016 ◽  
Vol 84 (12) ◽  
pp. 3458-3470 ◽  
Author(s):  
Mike Khan ◽  
Jerome S. Harms ◽  
Fernanda M. Marim ◽  
Leah Armon ◽  
Cherisse L. Hall ◽  
...  
Keyword(s):  
Immune Response ◽  
Second Messenger ◽  
Host Immune Response ◽  
Vital Role ◽  
Host Cells ◽  
Content Type ◽  
Type Iv

Brucella species are facultative intracellular bacteria that cause brucellosis, a chronic debilitating disease significantly impacting global health and prosperity. Much remains to be learned about how Brucella spp. succeed in sabotaging immune host cells and how Brucella spp. respond to environmental challenges. Multiple types of bacteria employ the prokaryotic second messenger cyclic di-GMP (c-di-GMP) to coordinate responses to shifting environments. To determine the role of c-di-GMP in Brucella physiology and in shaping host- Brucella interactions, we utilized c-di-GMP regulatory enzyme deletion mutants. Our results show that a Δ bpdA phosphodiesterase mutant producing excess c-di-GMP displays marked attenuation in vitro and in vivo during later infections. Although c-di-GMP is known to stimulate the innate sensor STING, surprisingly, the Δ bpdA mutant induced a weaker host immune response than did wild-type Brucella or the low-c-di-GMP guanylate cyclase Δ cgsB mutant. Proteomics analysis revealed that c-di-GMP regulates several processes critical for virulence, including cell wall and biofilm formation, nutrient acquisition, and the type IV secretion system. Finally, Δ bpdA mutants exhibited altered morphology and were hypersensitive to nutrient-limiting conditions. In summary, our results indicate a vital role for c-di-GMP in allowing Brucella to successfully navigate stressful and shifting environments to establish intracellular infection.

scholarly journals Anti-Trypanosoma cruzieffects of cyclosporin A derivatives: possible role of a P-glycoprotein and parasite cyclophilins

Parasitology ◽  
2007 ◽  
Vol 135 (2) ◽  
pp. 217-228 ◽  
Author(s):  
J. BÚA ◽  
L. E. FICHERA ◽  
A. G. FUCHS ◽  
M. POTENZA ◽  
M. DUBIN ◽  
...  
Keyword(s):  
Cyclosporin A ◽  
Mammalian Cells ◽  
Isomerase Activity ◽  
P Glycoprotein ◽  
Demethylase Activity ◽  
Target Molecules ◽  
Intracellular Amastigote

SUMMARYCyclophilins are target molecules for cyclosporin A (CsA), an immunosuppressive antimicrobial drug. We have previously reported thein vitroanti-Trypanosoma cruziactivity of H-7-94 and F-7-62 non-immunosuppressive CsA analogues. In this work, we continue the study of the parasiticidal effect of H-7-94 and F-7-62 CsA analoguesin vitroandin vivoand we analyse 3 new CsA derivatives: MeIle-4-CsA (NIM 811), MeVal-4-CsA (MeVal-4) and D-MeAla-3-EtVal-4-CsA, (EtVal-4). The most efficient anti-T. cruzieffect was observed with H-7-94, F-7-62 and MeVal-4 CsA analogues evidenced as inhibition of epimastigote proliferation, trypomastigote penetration, intracellular amastigote development andin vivo T. cruziinfection. This trypanocidal activity could be due to inhibition of the peptidyl prolylcis-transisomerase activity on theT. cruzirecombinant cyclophilins tested. Furthermore, CsA and F-7-62 derivative inhibited the efflux of rhodamine 123 fromT. cruziepimastigotes, suggesting an interference with a P-glycoprotein activity. Moreover, H-7-94 and F-7-62 CsA analogues were not toxic as shown by cell viability and by aminopyrine-N-demethylase activity on mammalian cells. Our results show that H-7-94, F-7-62 and MeVal-4 CsA analogues expressed the highest inhibiting effects onT. cruzi, being promissory parasiticidal drugs worthy of further studies.

Testing of Schefflera vinosa extract in mammalian cells in vitro and in vivo for potential toxicity, genetic damage, and role of oxidation

2016 ◽  
Vol 79 (24) ◽  
pp. 1201-1210 ◽  
Author(s):  
Natália Helen Ferreira ◽  
Kelly Jacqueline Barbosa de Andrade ◽  
Luís Fernando Leandro ◽  
Nathália Oliveira Acésio ◽  
Suzana Amorim Mendes ◽  
...  
Keyword(s):  
Mammalian Cells ◽  
Genetic Damage ◽  
Potential Toxicity

scholarly journals Role of Unc104/KIF1-related Motor Proteins in Mitochondrial Transport in Neurospora crassa

2005 ◽  
Vol 16 (1) ◽  
pp. 153-161 ◽  
Author(s):  
Florian Fuchs ◽  
Benedikt Westermann
Keyword(s):  
Neurospora Crassa ◽  
Mammalian Cells ◽  
Transport Systems ◽  
Eukaryotic Cells ◽  
Mitochondrial Transport ◽  
Specific Subgroup ◽  
Mitochondrial Motility

Eukaryotic cells use diverse cytoskeleton-dependent machineries to control inheritance and intracellular positioning of mitochondria. In particular, microtubules play a major role in mitochondrial motility in the filamentous fungus Neurospora crassa and in mammalian cells. We examined the role of two novel Unc104/KIF1-related members of the kinesin family, Nkin2 and Nkin3, in mitochondrial motility in Neurospora. The Nkin2 protein is required for mitochondrial interactions with microtubules in vitro. Mutant hyphae lacking Nkin2 show mitochondrial motility defects in vivo early after germination of conidiospores. Nkin3, a member of a unique fungal-specific subgroup of small Unc104/KIF1-related proteins, is not associated with mitochondria in wild-type cells. However, it is highly expressed and recruited to mitochondria in Δnkin-2 mutants. Mitochondria lacking Nkin2 require Nkin3 for binding to microtubules in vitro, and mitochondrial motility defects in Δnkin-2 mutants disappear with up-regulation of Nkin3 in vivo. We propose that mitochondrial transport is mediated by Nkin2 in Neurospora, and organelle motility defects in Δnkin-2 mutants are rescued by Nkin3. Apparently, a highly versatile complement of organelle motors allows the cell to efficiently respond to exogenous challenges, a process that might also account for the great variety of different mitochondrial transport systems that have evolved in eukaryotic cells.

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